Throughout this application, various publications are referenced in parenthesis by number. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosure of these publications is hereby incorporated by reference into this application to describe more fully the art to which this invention pertains.
Neuropeptides are small peptides originating from large precursor proteins synthesized by peptidergic neurons and endocrine/paracrine cells. They hold promise for treatment of neurological, psychiatric, and endocrine disorders (46). Often the precursors contain multiple biologically active peptides. There is great diversity of neuropeptides in the brain caused by alternative splicing of primary gene transcripts and differential precursor processing. The neuropeptide receptors serve to discriminate between ligands and to activate the appropriate signals.
Neuropeptide Y (NPY), a 36-amino acid peptide, is the most abundant neuropeptide to be identified in mammalian brain. NPY is an important regulator in both the central and peripheral nervous systems (47) and influences a diverse range of physiological parameters, including effects on psychomotor activity, food intake, central endocrine secretion, and vasoactivity in the cardiovascular system. High concentrations of NPY are found in the sympathetic nerves supplying the coronary, cerebral, and renal vasculature and have contributed to vasoconstriction. NPY binding sites have been identified in a variety of tissues, including spleen (48), intestinal membranes, brain (49), aortic smooth muscle (50), kidney, testis, and placenta (2). In addition, binding sites have been reported in a number of rat and human cell lines (e.g. Y1 in SK-N-MC, MC-IXC, CHP-212, and PC12 cells; Y2 in SK-N-Be(2), CHP-234, and SMS-MSN) (51,5).
Neuropeptide Y (NPY) receptor pharmacology is currently defined by structure activity relationships within the pancreatic polypeptide family (1, 2). This family includes NPY, which is synthesized primarily in neurons; peptide YY (PYY), which is synthesized primarily by endocrine cells in the gut; and pancreatic polypeptide (PP), which is synthesized primarily by endocrine cells in the pancreas. These 36 amino acid peptides have a compact helical structure involving a "PP-fold" in the middle of the peptide. Specific features include a polyproline helix in residues 1 through 8, a .beta.-turn in residues 9 through 14, an .alpha.-helix in residues 15 through 30, an outward-projecting C-terminus in residues 30 through 36, and a carboxyl terminal amide which appears to be critical for biological activity (3). The peptides have been used to define at least four receptor subtypes known as Y1, Y2, Y3, and PP. Y1 receptor recognition by NPY involves both N- and C-terminal regions of the peptide; exchange of Gln.sup.34 with Pro.sup.34 is fairly well tolerated (3, 4, 5). Y2 receptor recognition by NPY depends primarily upon the four C-terminal residues of the peptide (Arg.sup.33 -Gln.sup.34 -Arg.sup.35 -Tyr36-NH.sub.2) preceded by an amphipathic .alpha.-helix (3, 6, 7); exchange of Gln.sup.34 with Pro.sup.34 is not well tolerated (4, 5). Y3 receptor recognition is characterized by a strong preference for NPY over PYY (8). Exchange of Gln.sup.34 in NPY with Pro.sup.34 is reasonably well tolerated by the Y3 receptor but PP, which also contains Pro.sup.34, does not bind well (8). The PP receptor is reported to bind tightly to PP, less so to [Leu.sup.31,Pro.sup.34 ]NPY, and even less so to NPY (3, 9). The only NPY receptor which has been cloned to date is the Y1 receptor gene, from mouse (12), rat (52), and human (10). One of the key pharmacological features which distinguish Y1 and Y2 is the fact that the Y1 receptor (and not the Y2 receptor) responds to an analog of NPY modified at residues 31 and 34 ([Leu31,Pro34]NPY), whereas the Y2 receptor (and not the Y1 receptor) has high affinity for the NPY peptide carboxyl-terminal fragment NPY-(13-36) (53,4).
Receptor genes for the other two structurally related peptides, peptide YY (PYY) and pancreatic polypeptide (PP), also have not been cloned. Peptide YY occurs mainly in endocrine cells in the lower gastrointestinal tract (54). Receptors for PYY were first described in the rat small intestine (55). This receptor has been defined as PYY-preferring because it displays a 5-10 fold higher affinity for PYY than for NPY (55, 56). Recently, a cell line, PKSV-PCT, derived from the proximal tubules of kidneys, has been described to express receptors for PYY (57).
In the last few years only the rat and human Y1 cDNAs have been cloned (10, 11). This success was based on identifying the randomly cloned FC5 "orphan receptor" (12). We now report the isolation by expression cloning of a human hippocampal Y2 cDNA clone and two rat Y2 clones and their pharmacological characterization.